U.S. patent number 11,261,477 [Application Number 16/923,986] was granted by the patent office on 2022-03-01 for glycosylation modification of bioactive compounds and drugs by plant glycosyltransferases (ugts).
This patent grant is currently assigned to UNIVERSITY OF NORTH TEXAS. The grantee listed for this patent is UNIVERSITY OF NORTH TEXAS. Invention is credited to Xiaoqiang Wang.
United States Patent |
11,261,477 |
Wang |
March 1, 2022 |
Glycosylation modification of bioactive compounds and drugs by
plant glycosyltransferases (UGTs)
Abstract
In alternative embodiments, provided are methods for the
glycosylation modification of bioactive compounds and drugs using
isolated, recombinant or genetically modified uridine diphosphate
glycosyl-transferases (UGTs). In alternative embodiments, provided
are methods for modifying UGTs to generate recombinant UGTs with
altered donor and/or acceptor specificities. In alternative
embodiments, provided are methods for screening for recombinantly
engineered UGTs with new or altered properties, for example, for
new or altered donor and/or acceptor specificities, where in
alternative embodiments the screening comprise use of bacterial,
yeast or baculovirus expression system.
Inventors: |
Wang; Xiaoqiang (Denton,
TX) |
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF NORTH TEXAS |
Denton |
TX |
US |
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Assignee: |
UNIVERSITY OF NORTH TEXAS
(Denton, TX)
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Family
ID: |
1000006140796 |
Appl.
No.: |
16/923,986 |
Filed: |
July 8, 2020 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20210010056 A1 |
Jan 14, 2021 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62873620 |
Jul 12, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/48 (20130101); C12N 9/1051 (20130101); G01N
2333/91102 (20130101); C12Y 204/01017 (20130101) |
Current International
Class: |
C12N
9/10 (20060101); C12Q 1/48 (20060101) |
Other References
Kren et al., "Glycosides in Medicine: "The Role of Glycosidic
Residue in Biological Activity"" Current Medicinal Chemistry 2001,
v 8, p. 1303-1328. cited by applicant .
Bowles et al., "Glycosyltransferases: managers of small molecules"
Current Opinion in Plant Biology 2005, v 8, p. 254-263. cited by
applicant .
Shimoda et al., "Synthesis of Oligosaccharides of Genistein and
Quercetin as Potential Anti-inflammatory Agents" Chemistry Letters,
2008, v 37, n 8, p. 876-877. cited by applicant .
Wang, "Structure, mechanism and engineering of plant natural
product glycosyltransferases" FEBS Letters, 2019, 583, p.
3303-3309. cited by applicant .
Shao et al., "Crystal Structures of a Multifunctional
Triterpene/Flavonoid Glycosyltransferase from Medicago truncatula"
The Plant Cell, Nov. 2005. cited by applicant.
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Primary Examiner: Saidha; Tekchand
Attorney, Agent or Firm: Greer, Burn & Crain, Ltd.
Einhorn; Gregory P.
Parent Case Text
RELATED APPLICATIONS
This U.S. utility patent application claims the benefit of priority
to U.S. Provisional Patent Application Ser. No. 62/873,620 filed
Jul. 12, 2019. The aforementioned application is expressly
incorporated herein by reference in its entirety and for all
purposes.
Claims
What is claimed is:
1. A method for identifying or screening for a recombinant or
genetically modified uridine diphosphate glycosyl-transferase (UGT)
having a modified sequence such that the modification of the UGT
results in the glycosylation of or adding a sugar moiety to an
otherwise unglycosylated bioactive compound or target drug, or
results in generating a modified glycosylation of a bioactive
compound or a target drug by adding a sugar moiety, the method
comprising: (a) providing or having provided a recombinant or
genetically modified UGT comprising a M. truncatula uridine
diphosphate glycosyl-transferase (UGT) UGT71G1, wherein the
expressed recombinant or genetically modified UGT has an altered or
new donor and/or acceptor specificity, (b) providing or having
provided an acceptor molecule, (c) expressing the recombinant or
genetically modified UGT in an expression system, (d) contacting
the recombinant or genetically modified UGT with a bioactive
compound or a target drug in the expression system, and (e)
screening for the generation of a UGT that results in the
glycosylation of an otherwise unglycosylated acceptor molecule, or
results in generating a modified glycosylation of the acceptor
molecule.
2. The method of claim 1, wherein the recombinant or genetically
modified UGT uses UDP-glucose, UDP-glucuronic acid and/or
UDP-rhamnose as a donor, thereby adding a glucose, glucuronic acid
and/or a rhamnose sugar moiety to the acceptor molecule.
3. The method of claim 1, wherein the recombinant or genetically
modified UGT uses a drug or a small molecule as an acceptor, or the
acceptor molecule comprises a drug or a small molecule.
4. The method of claim 1, wherein the acceptor-binding pocket of
the recombinant or genetically modified UGT is modified.
5. The method of claim 1, wherein the expression system is a
cell-based expression system or an in vitro expression system.
6. The method of claim 5, wherein the cell-based expression system
comprises a bacterial, a yeast, a baculovirus or a mammalian
cell-based expression system.
7. The method of claim 1, wherein the acceptor molecule is a
terpene, a terpenoid, a flavonoid, an isoflavonoid or a natural
product.
8. The method of claim 7, wherein the natural product, terpene,
terpenoid, flavonoid or isoflavonoid is: ursolic acid,
liquiritigenin, 3-Carene; 3,7(11)-Eudesmadiene; 4-Carvomenthenol;
4-Thujanol; alpha-Bergamotene; alpha-Bisabolol, (+)-;
alpha-Bulnesene; alpha-Cedrene; alpha-Guaiene; alpha-Ocimene,
(3E)-; alpha-Phellandrene; alpha-Pinene; alpha-Terpinene;
Aromadendrene; beta-Caryophyllene; beta-Elemene; beta-Farnesene,
(6E)-; beta-Ocimene; beta-Pinene; beta-Thujene; Cannabidiol;
Cannabigerolic Acid; Carvone, (-)-; Caryophyllene Oxide; Cedrol;
cis-2-Pinanol; cis-beta-ocimene; cis-Nerolidol; Citronellol;
d-Limonene; delta8-THC; Dronabinol; Eucalyptol; Fenchone; Fenchol;
gamma-Elemene; gamma-Terpinene; Geraniol; Geranyl Acetate;
Germacrene B; Guaiol; Humulene; (-)-; Isopulegol; Limonene;
Linalool; Menthol; Myrcene; Nerol; Nerolidol; p-Cymene; Phytol;
Pulegone; Sabinene; Sabinene Hydrate; Terpineol; Terpinolene;
Valencene; (-)-Terpinen-4-ol, (-)-Terpinen-4-ol, d-limonene
linalool, 1,8-cineole (eucalyptol), .alpha.-pinene, terpineol-4-ol,
p-cymene, .DELTA.-3-carene, .beta.-sitosterol, .beta.-myrcene,
.beta.-caryophyllene, cannflavin A, apigenin, quercetin, pulegone,
borneol; isoborneol; camphene; camphor; delta-3-carene;
beta-caryophyllene; caryophyllene oxide; alpha-cedrene;
beta-eudesmol; fenchyl alcohol; geraniol; guaiol; alpha-humulene;
limonene; linalool; menthol; myrcene; nerol; ocimene;
trans-ocimene; alpha-phellandrene; alpha-pinene; beta-pinene;
sabinene; alpha-terpinene; alpha-terpineol; terpinolene;
alpha-guaiene; elemene; farnesene; germacrene B; guaia-1(10),
11-diene; trans-2-pinanol; selina-3,7(11)-diene;
eudesm-7(11)-en-4-ol; valencene; 7,8-dihydroionone, Acetanisole,
Acetic Acid, Acetyl Cedrene, Anethole, Anisole, Benzaldehyde,
Bergamotene (.alpha.-cis-Bergamotene) (.alpha.-trans-Bergamotene),
Bisabolol (.beta.-Bisabolol), Borneol, Butanoic/Butyric Acid,
Cadinene (.alpha.-Cadinene) (.gamma.-Cadinene), Cafestol, Caffeic
acid, Camphene, Camphor, Capsaicin, Carene (.DELTA.-3-Carene),
Carotene, Carvacrol, Carvone, Dextro-Carvone, Laevo-Carvone,
Caryophyllene (.beta.-Caryophyllene), Caryophyllene oxide,
Castoreum Absolute, Cedrene (.alpha.-Cedrene) (.beta.-Cedrene),
Cedrene Epoxide (.alpha.-Cedrene Epoxide), Cedrol, Cembrene,
Chlorogenic Acid, Cinnamaldehyde (.alpha.-amyl-Cinnamaldehyde)
(.alpha.-hexyl-Cinnamaldehyde), Cinnamic Acid, Cinnamyl Alcohol,
Citronellal, Citronellol, Cryptone, Curcumene (.alpha.-Curcumene)
(.gamma.-Curcumene), Decanal, Dehydrovomifoliol, Diallyl Disulfide,
Dihydroactinidiolide, Dimethyl Disulfide, Eicosane/lcosane, Elemene
(.beta.-Elemene), Estragole, Ethyl acetate, Ethyl Cinnamate, Ethyl
maltol, Eucalyptol/1,8-Cineole, Eudesmol (.alpha.-Eudesmol)
(.beta.-Eudesmol) (.gamma.-Eudesmol), Eugenol, Euphol, Farnesene,
Farnesol, Fenchol (.beta.-Fenchol), Fenchone, Geraniol, Geranyl
acetate, Germacrenes, Germacrene B, Guaia-1 (10), 1 1-diene,
Guaiacol, Guaiene (.alpha.-Guaiene), Gurjunene (.alpha.-Gurjunene),
Herniarin, Hexanaldehyde, Hexanoic Acid, Humulene
(.alpha.-Humulene) (.beta.-Humulene), lonol (3-oxo-.alpha.-ionol)
(.beta.-lonol), Ionone (.alpha.-Ionone) (.beta.-Ionone), Ipsdienol,
Isoamyl acetate, Isoamyl Alcohol, Isoamyl Formate, Isoborneol,
Isomyrcenol, Isopulegol, Isovaleric Acid, Isoprene, Kahweol,
Lavandulol, Limonene, y-Linolenic Acid, Linalool, Longifolene,
a-Longipinene, Lycopene, Menthol, Methyl butyrate,
3-Mercapto-2-Methylpentanal, Mercaptan/Thiols,
.beta.-Mercaptoethanol, Mercaptoacetic Acid, AIM Mercaptan, Benzyl
Mercaptan, Butyl Mercaptan, Ethyl Mercaptan, Methyl Mercaptan,
Furfuryl Mercaptan, Ethylene Mercaptan, Propyl Mercaptan, Thenyl
Mercaptan, Methyl Salicylate, Methylbutenol,
Methyl-2-Methylvalerate, Methyl Thiobutyrate, Myrcene
(.beta.-Myrcene), .gamma.-Muurolene, Nepetalactone, Nerol,
Nerolidol, Neryl acetate, Nonanaldehyde, Nonanoic Acid, Ocimene,
Octanal, Octanoic Acid, p-cymene, pentyl butyrate, phellandrene,
phenylacetaldehyde, phenylethanethiol, Phenylacetic Acid, Phytol,
Pinene, .beta.-Pinene, propanethiol, Pristimerin, Pulegone,
Retinol, Rutin, Sabinene, Sabinene Hydrate, cis-Sabinene Hydrate,
trans-Sabinene Hydrate, Safranal, .alpha.-Selinene,
.alpha.-Sinensal, .beta.-Sinensal, .beta.-Sitosterol, Squalene,
Taxadiene, Terpin hydrate, Terpineol, Terpine-4-ol,
.alpha.-Terpinene, y-Terpinene, Terpinolene, Thiophenol, Thujone,
Thymol, .alpha.-Tocopherol, Tonka Undecanone, Undecanal,
Valeraldehyde/Pentanal, Verdoxan, .alpha.-Ylangene, Umbelliferone,
Vanillin, a phenolic acid, a stilbenoid, a dihydroflavonol, an
anthocyanin, an anthocyanidin, a polyphenol, a tannin, a flavone,
flavan-3-ol, flavan-4-ol, flavan-3,4-diol flavonol, a stilbenoid, a
phytochemicals, an antioxidant, a homoisoflavonoid, a
phenylpropanoid, a phloroglucinol coumarin, a phenolic acid, a
naphthodianthrone, a steroid glycoside, a bioflavonoid, an
isoflavonoid, a neoflavonoid, adenosine, Adhyperforin,
amentoflavone, Anandamide, Apigenin, Cannaflavin B, Catechin (C),
Catechin 3-gallate (Cg), Chlorogenic acid, cichoric acid, caftaric
acid, Daidzein, Delphinidin, Eleutherosides, epicatechin 3-gallate
(ECg), Epicatechins, Epicatechin, epigallocatechin, myricetin,
Oxalic acid, Pelargonidin, Tannin, Theaflavin-3-gallate, Theanine,
Theobromine, Theophylline, Tryptophan, Tyramine, Xanthine,
Caffeine, Cannaflavin A, Cannaflavin B, Catechin (C), Catechin
3-gallate (Cg), Epicatechin 3-gallate (ECg), Epicatechins
(Epicatechin (EC)), epigallocatechin, Epigallocatechin (EGC),
Epigallocatechin 3-gallate (EGCg), Gallocatechin (GC),
Gallocatechin 3-gallate (GCg)), Gamma amino butyric acid,
Genistein, Ginkgo biloba, Ginsenosides, Quercetin, Quercitrin or
Rutin.
9. The method of claim 1, wherein the recombinant or genetically
modified UGT can catalyze a reverse reaction to remove a sugar
moiety from a glycosylated bioactive compound or a target drug.
10. The method of claim 1, wherein the recombinant or genetically
modified UGT is glycosyltransferase M. truncatula UGT71G1has as
substrates (can glycosylate) quercetin, genistein, biochanin A,
hederagenin, and SN-38 (active metabolite of anti-cancer drug
CPT-11 or irinotecan), and optionally (iso)flavonoids quercetin and
genistein as in vitro substrates.
Description
TECHNICAL FIELD
This invention generally relates to pharmaceutical drug development
and small molecule glycosylation processes. In alternative
embodiments, provided are methods for the glycosylation
modification of bioactive compounds and drugs using isolated,
recombinant or genetically modified uridine diphosphate
glycosyl-transferases (UGTs). In alternative embodiments, provided
are methods for modifying UGTs to generate recombinant UGTs with
altered donor and/or acceptor specificities. In alternative
embodiments, provided are methods for screening for recombinantly
engineered UGTs with new or altered properties, for example, for
new or altered donor and/or acceptor specificities, where in
alternative embodiments the screening comprise use of bacterial,
yeast or baculovirus expression systems.
BACKGROUND
Glycosylation is a biological process that improves bioavailability
and therefore pharmacological activities. The main implication of
glycosylation is in drug discovery. Uridine diphosphate (UDP)
glycosyltransferases (UGTs) are key components in the glycosylation
process. They can be used for modifying small molecular drugs with
various sugars.
A major issue in drug modification and discovery is that it is
difficult to perform targeted glycosylation of specific drug
scaffolds with current conventional synthetic approaches. Different
UGTs have unique sequences and specific templates.
The UGT UGT71G1 is a multifunctional glycosyltransferase and can
glycosylate terpenoids such as hederagenin, flavonoids such as
quercetin, and SN-38, an active metabolite of the anti-cancer drug
CPT-11 (irinotecan). Terpenoids have been proven to benefit human
health, where some can fight malaria, some can be used to treat
cancer.
SUMMARY
In alternative embodiments, provided are methods for designing and
generating recombinant or genetically modified uridine diphosphate
glycosyl-transferases (UGTs) with modified sequences and
compositions complementing target drugs, the method comprising:
expressing a recombinant or genetically modified UGT in an E. coli,
a yeast or a baculovirus expression system.
In alternative embodiments of methods as provided herein, the
expressed recombinant or genetically modified UGT has an altered or
new donor and/or acceptor specificity, and optionally the
recombinant or genetically modified UGT uses UDP-glucuronic acid or
UDP-rhamnose as a donor.
In alternative embodiments of methods as provided herein, the
expressed recombinant or genetically modified UGT has an altered or
new donor and/or acceptor specificity, and optionally the
recombinant or genetically modified UGT uses a drug or small
molecule as an acceptor, and optionally the acceptor-binding pocket
of the UGT is modified.
In alternative embodiments of methods as provided herein, the
expressed recombinant or genetically modified UGT has an altered or
new donor and/or acceptor specificity, and the method further
comprises screening for the new or altered UGT donor and/or
acceptor specificity in a bacterial, yeast or baculovirus
expression system.
In alternative embodiments, provided are methods for identifying or
screening for a recombinant or genetically modified uridine
diphosphate glycosyl-transferase (UGT) having a modified sequence
such that the modification of the UGT results in the glycosylation
of or adding a sugar moiety to an otherwise unglycosylated
bioactive compound or target drug, or results in generating a
modified glycosylation of a bioactive compound or a target drug by
adding a sugar moiety, the method comprising:
(a) providing or having provided a recombinant or genetically
modified UGT, wherein the expressed recombinant or genetically
modified UGT has an altered or new donor and/or acceptor
specificity,
(b) providing or having provided an acceptor molecule,
(c) expressing the recombinant or genetically modified UGT in an
expression system,
(d) contacting the recombinant or genetically modified UGT with a
bioactive compound or a target drug in the expression system,
and
(e) screening for the generation of a UGT that results in the
glycosylation of an otherwise unglycosylated acceptor molecule, or
results in generating a modified glycosylation of the acceptor
molecule.
In alternative embodiments of methods as provided herein: the
recombinant or genetically modified UGT uses UDP-glucose,
UDP-glucuronic acid and/or UDP-rhamnose as a donor, thereby adding
a glucose, glucuronic acid and/or a rhamnose sugar moiety to the
acceptor molecule; the recombinant or genetically modified UGT uses
a drug or a small molecule as an acceptor, or the acceptor molecule
comprises a drug or a small molecule the acceptor-binding pocket of
the recombinant or genetically modified UGT is modified; the
expression system is a cell-based expression system or an in vitro
expression system, and the cell-based expression system can
comprise a bacterial, a yeast, a baculovirus, or a mammalian
expression system; the UGT is UGT71G1, optionally a Medicago
UGT71G1; the acceptor molecule is a terpene, a terpenoid, a
flavonoid, an isoflavonoid or a natural product, and optionally the
natural product, terpene, terpenoid, flavonoid or isoflavonoid is:
ursolic acid, liquiritigenin, 3-Carene; 3,7(11)-Eudesmadiene;
4-Carvomenthenol; 4-Thujanol; alpha-Bergamotene; alpha-Bisabolol,
(+)-; alpha-Bulnesene; alpha-Cedrene; alpha-Guaiene; alpha-Ocimene,
(3E)-; alpha-Phellandrene; alpha-Pinene; alpha-Terpinene;
Aromadendrene; beta-Caryophyllene; beta-Elemene; beta-Farnesene,
(6E)-; beta-Ocimene; beta-Pinene; beta-Thujene; Cannabidiol;
Cannabigerolic Acid; Carvone, (-)-; Caryophyllene Oxide; Cedrol;
cis-2-Pinanol; cis-beta-ocimene; cis-Nerolidol; Citronellol;
d-Limonene; delta8-THC; Dronabinol; Eucalyptol; Fenchone; Fenchol;
gamma-Elemene; gamma-Terpinene; Geraniol; Geranyl Acetate;
Germacrene B; Guaiol; Humulene; (-)-; Isopulegol; Limonene;
Linalool; Menthol; Myrcene; Nerol; Nerolidol; p-Cymene; Phytol;
Pulegone; Sabinene; Sabinene Hydrate; Terpineol; Terpinolene;
Valencene; (-)-Terpinen-4-ol, (-)-Terpinen-4-ol, d-limonene
linalool, 1,8-cineole (eucalyptol), .alpha.-pinene, terpineol-4-ol,
p-cymene, .DELTA.-3-carene, .beta.-sitosterol, .beta.-myrcene,
.beta.-caryophyllene, cannflavin A, apigenin, quercetin, pulegone,
borneol; isoborneol; camphene; camphor; delta-3-carene;
beta-caryophyllene; caryophyllene oxide; alpha-cedrene;
beta-eudesmol; fenchyl alcohol; geraniol; guaiol; alpha-humulene;
limonene; linalool; menthol; myrcene; nerol; ocimene;
trans-ocimene; alpha-phellandrene; alpha-pinene; beta-pinene;
sabinene; alpha-terpinene; alpha-terpineol; terpinolene;
alpha-guaiene; elemene; farnesene; germacrene B; guaia-1(10),
11-diene; trans-2-pinanol; selina-3,7(11)-diene;
eudesm-7(11)-en-4-ol; valencene; 7,8-dihydroionone, Acetanisole,
Acetic Acid, Acetyl Cedrene, Anethole, Anisole, Benzaldehyde,
Bergamotene (.alpha.-cis-Bergamotene) (.alpha.-trans-Bergamotene),
Bisabolol (.beta.-Bisabolol), Borneol, Butanoic/Butyric Acid,
Cadinene (.alpha.-Cadinene) (.gamma.-Cadinene), Cafestol, Caffeic
acid, Camphene, Camphor, Capsaicin, Carene (.DELTA.-3-Carene),
Carotene, Carvacrol, Carvone, Dextro-Carvone, Laevo-Carvone,
Caryophyllene (.beta.-Caryophyllene), Caryophyllene oxide,
Castoreum Absolute, Cedrene (.alpha.-Cedrene) (.beta.-Cedrene),
Cedrene Epoxide (.alpha.-Cedrene Epoxide), Cedrol, Cembrene,
Chlorogenic Acid, Cinnamaldehyde (.alpha.-amyl-Cinnamaldehyde)
(.alpha.-hexyl-Cinnamaldehyde), Cinnamic Acid, Cinnamyl Alcohol,
Citronellal, Citronellol, Cryptone, Curcumene (.alpha.-Curcumene)
(.gamma.-Curcumene), Decanal, Dehydrovomifoliol, Diallyl Disulfide,
Dihydroactinidiolide, Dimethyl Disulfide, Eicosane/lcosane, Elemene
(.beta.-Elemene), Estragole, Ethyl acetate, Ethyl Cinnamate, Ethyl
maltol, Eucalyptol/1,8-Cineole, Eudesmol (.alpha.-Eudesmol)
(.beta.-Eudesmol) (.gamma.-Eudesmol), Eugenol, Euphol, Farnesene,
Farnesol, Fenchol (.beta.-Fenchol), Fenchone, Geraniol, Geranyl
acetate, Germacrenes, Germacrene B, Guaia-1 (10), 1 1-diene,
Guaiacol, Guaiene (.alpha.-Guaiene), Gurjunene (.alpha.-Gurjunene),
Herniarin, Hexanaldehyde, Hexanoic Acid, Humulene
(.alpha.-Humulene) (.beta.-Humulene), lonol (3-oxo-.alpha.-ionol)
(.beta.-Io.eta.oI), lonone (.alpha.-lonone) (.beta.-lonone),
Ipsdienol, Isoamyl acetate, Isoamyl Alcohol, Isoamyl Formate,
Isoborneol, Isomyrcenol, Isopulegol, Isovaleric Acid, Isoprene,
Kahweol, Lavandulol, Limonene, .gamma.-Linolenic Acid, Linalool,
Longifolene, .alpha.-Longipinene, Lycopene, Menthol, Methyl
butyrate, 3-Mercapto-2-Methylpentanal, Mercaptan/Thiols,
.beta.-Mercaptoethanol, Mercaptoacetic Acid, AIM Mercaptan, Benzyl
Mercaptan, Butyl Mercaptan, Ethyl Mercaptan, Methyl Mercaptan,
Furfuryl Mercaptan, Ethylene Mercaptan, Propyl Mercaptan, Thenyl
Mercaptan, Methyl Salicylate, Methylbutenol,
Methyl-2-Methylvalerate, Methyl Thiobutyrate, Myrcene
(.beta.-Myrcene), .gamma.-Muurolene, Nepetalactone, Nerol,
Nerolidol, Neryl acetate, Nonanaldehyde, Nonanoic Acid, Ocimene,
Octanal, Octanoic Acid, p-cymene, pentyl butyrate, phellandrene,
phenylacetaldehyde, phenylethanethiol, Phenylacetic Acid, Phytol,
Pinene, .beta.-Pinene, propanethiol, Pristimerin, Pulegone,
Retinol, Rutin, Sabinene, Sabinene Hydrate, cis-Sabinene Hydrate,
trans-Sabinene Hydrate, Safranal, .alpha.-Selinene,
.alpha.-Sinensal, .beta.-Sinensal, .beta.-Sitosterol, Squalene,
Taxadiene, Terpin hydrate, Terpineol, Terpine-4-ol,
.alpha.-Terpinene, .gamma.-Terpinene, Terpinolene, Thiophenol,
Thujone, Thymol, .alpha.-Tocopherol, Tonka Undecanone, Undecanal,
Valeraldehyde/Pentanal, Verdoxan, .alpha.-Ylangene, Umbelliferone,
Vanillin, a phenolic acid, a stilbenoid, a dihydroflavonol, an
anthocyanin, an anthocyanidin, a polyphenol, a tannin, a flavone,
flavan-3-ol, flavan-4-ol, flavan-3,4-diol flavonol, a stilbenoid, a
phytochemicals, an antioxidant, a homoisoflavonoid, a
phenylpropanoid, a phloroglucinol coumarin, a phenolic acid, a
naphthodianthrone, a steroid glycoside, a bioflavonoid, an
isoflavonoid, a neoflavonoid, adenosine, Adhyperforin,
amentoflavone, Anandamide, Apigenin, Cannaflavin B, Catechin (C),
Catechin 3-gallate (Cg), Chlorogenic acid, cichoric acid, caftaric
acid, Daidzein, Delphinidin, Eleutherosides, epicatechin 3-gallate
(ECg), Epicatechins, Epicatechin, epigallocatechin, myricetin,
Oxalic acid, Pelargonidin, Tannin, Theaflavin-3-gallate, Theanine,
Theobromine, Theophylline, Tryptophan, Tyramine, Xanthine,
Caffeine, Cannaflavin A, Cannaflavin B, Catechin (C), Catechin
3-gallate (Cg), Epicatechin 3-gallate (ECg), Epicatechins
(Epicatechin (EC)), epigallocatechin, Epigallocatechin (EGC),
Epigallocatechin 3-gallate (EGCg), Gallocatechin (GC),
Gallocatechin 3-gallate (GCg)), Gamma amino butyric acid,
Genistein, Ginkgo biloba, Ginsenosides, Quercetin, Quercitrin or
Rutin; the recombinant or genetically modified UGT can catalyze a
reverse reaction to remove a sugar moiety from a glycosylated
bioactive compound or a target drug; the recombinant or genetically
modified UGT is glycosyl-transferase UGT78G1 from Medicago
truncatula, which can catalyze the glycosylation of isoflavonoids
and flavonoids, optionally the flavonols kaempferol and myricetin,
the isoflavone formononetin, and/or the anthocyanidins pelargonidin
and cyanidin; the recombinant or genetically modified UGT is
glycosyltransferase M. truncatula UGT72L1, which optionally can
produce epicatechin 3'-O-glucoside; the recombinant or genetically
modified UGT is glycosyltransferase M. truncatula UGT71G1, which
uses UDP-glucose as a sugar donor, and is a multifunctional
glycosyltransferase, and optionally has as substrates (can
glycosylate) quercetin, genistein, biochanin A, hederagenin, and
SN-38 (active metabolite of anti-cancer drug CPT-11 or irinotecan),
and optionally (iso)flavonoids quercetin and genistein as in vitro
substrates; the recombinant or genetically modified UGT is
glycosyl-transferase UGT85H2, which utilizes UDP-glucose as a
donor, and which is a multifunctional (iso)flavonoid
glycosyltransferase with activity toward several flavonoid-related
secondary metabolites, including isoflavones, flavonols, and
chalcone, and optionally has as substrates quercetin; genistein;
biochanin A, kaempferol, and isoliquiritigenin; the recombinant or
genetically modified UGT is glycosyl-transferase Medicago UGT78G1,
which utilizes UDP-glucose as a donor, which is an (iso)flavonoid
glycosyltransferase with broad activities on isoflavones,
optionally formononetin and flavonols, optionally kaempferol, and
optionally has activity for the anthocyanidins pelargonidin and
cyanidin; the recombinant or genetically modified UGT is UGT78G1,
which can catalyze a reverse reaction to remove a sugar moiety,
where the enzyme converts biochanin A 7-O-glucoside, genistein
7-O-glucoside, kaempferol 3-O-glucoside, and quercetin
3-O-glucoside into corresponding aglycones; and optionally UGT78G1
uses quercetin; genistein; Biochanin A, kaempferol; formononetin,
daidzein, apigenin, myricetin, pelargonidin and cyanidin as a
substrate; the recombinant or genetically modified UGT is
glycosyl-transferase UGT88D7, which utilizes UDP-glucuronic acid as
a donor, and optionally uses apigenin, baicalein, scutellarein,
kaempferol, quercetin and naringenin as a substrate; the
recombinant or genetically modified UGT is glycosyl-transferase
BpUGT94B1, which utilizes UDP-glucuronic acid as a donor, and
optionally uses cyanidin 3-O-glucoside and cyanidin
3-O-6'-O-malonylglucoside as a substrate; the recombinant or
genetically modified UGT is glycosyl-transferase UGT89C1, which
utilizes UDP-rhamnose as a donor, and optionally uses kaempferol,
kae3-O-glucoside and quercetin 3-O-glucoside as a substrate; the
recombinant or genetically modified UGT is glycosyltransferase rice
OsCGT, which utilizes UDP-glucose as a donor, and optionally uses
phloretin, 2-hydroxy-eriodictyol and 2-hydroxynaringenin as a
substrate; and/or the recombinant or genetically modified UGT is
glycosyl-transferase buckwheat FeCGTa (UGT708C1), which utilizes
UDP-glucose as a donor, and optionally uses phloretin,
2-hydroxyeriodictyol and 2-hydroxynaringenin as a substrate.
The details of one or more exemplary embodiments of the invention
are set forth in the accompanying drawings and the description
below. Other features, objects, and advantages of the invention
will be apparent from the description and drawings, and from the
claims.
All publications, patents, patent applications cited herein are
hereby expressly incorporated by reference for all purposes.
DESCRIPTION OF DRAWINGS
The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
The drawings set forth herein are illustrative of exemplary
embodiments provided herein and are not meant to limit the scope of
the invention as encompassed by the claims.
FIG. 1A-C illustrate images of crystal structures of three plant
UGTs:
FIG. 1A illustrates the structure of Medicago UGT71G1;
FIG. 1B illustrates the structure of donor binding site and
interaction between the donor molecule UDP-glucose and the enzyme;
a plant UGT specific PSPG signature motif is shown as a ribbon
model in yellow; the structure of UDP-glucose is shown as a
ball-and-stick model;
FIG. 1C illustrates structures comparing acceptor binding pockets
of several plant UGTs including UGT71G1 (dimgrey), UGT78G1 (cyan),
VvGT1 (blue), UGT85H2 (orange), and UGT72B1 (lightgrey); catalytic
residues are in the UGT71G1 structure; residue numbers in UGT71G1
are labeled, and the UGT72B1 unique long loop (approximately
residue 315) is also labeled;
as discussed in detail in Example 1, below.
FIG. 2 illustrates a substrate binding pocket of UGT88D7 docked
with epicatechin and UDP-glucuronic acid (UDP-GA), as discussed in
detail in Example 1, below.
Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
In alternative embodiments, provided are methods for the enzymatic
glycosylation modification of bioactive compounds and drugs using
isolated, recombinant or genetically modified uridine diphosphate
glycosyl-transferases (UGTs), where UDP-glucose, UDP-glucuronic
acid and/or UDP-rhamnose can be donor molecules, and the acceptor
molecule is any small molecule, for example, a terpenoid (e.g.,
ursolic acid) or a flavonoid (e.g., liquiritigenin) or an
isoflavonoid (e.g, genistein). In alternative embodiments, some UGT
glycosyltransferases or their mutants also can catalyze a reverse
reaction to remove a sugar moiety from a glycosides.
In alternative embodiments, provided are methods for generating
chimera UGT enzymes with a modified or genetically engineered
acceptor substrate binding pocket complementing with target drug
compounds for glycosylation of various drugs with UDP-glucose,
UDP-glucuronic acid and/or UDP-rhamnose. For example, the acceptor
binding site modification can be based on the UGT78G1 structure,
where the acceptor-binding pocket is formed by several helices and
loops, and is mainly hydrophobic with many aromatic and other
hydrophobic residues, including Phe21, Phe93, Phe202, and Phe374;
and has several charged residues in the acceptor-binding pocket,
including His26, His155, and Asp376; and His26 acts as a catalytic
residue for UGT78G1, and the corresponding histidine acts as a
catalytic residue and general base for UGT71G1, VvGT1, and other
UGTs; also, an acidic residue, Asp124, forms a hydrogen bond with
His26 and also plays an essential role in catalysis.
In alternative embodiments, provided are methods for generating
chimera UGT enzymes with a modified or genetically engineered donor
binding sites. The donor binding site modification can be based on
the UGT78G1 structure, where the UDP donor molecule interacts with
a PSPG motif (Trp334-Gln377) in the C-terminal domain of the
enzyme; and a uracil ring of the UDP forms parallel .pi.-stacking
interactions with the indole ring of Trp334 and forms hydrogen
bonds with Ala335 (via its main-chain N and C atoms); and the
ribose ring of the UDP molecule forms hydrogen bonds with Gln337
and Glu360; and the a-phosphate group contacts Asn356 and Ser357;
and the .beta.-phosphate forms hydrogen bonds with His352.
Additionally, Ser308 forms a hydrogen bond with the uracil ring of
the UDP molecule, and Thr25 and Ser282 interact with its
.beta.-phosphate group.
In alternative embodiments, the UGT is the glycosyltransferase
UGT78G1 from Medicago truncatula, which an catalyze the
glycosylation of various (iso)flavonoids such as the flavonols
kaempferol and myricetin, the isoflavone formononetin, and the
anthocyanidins pelargonidin and cyanidin.
In alternative embodiments, the UGT is the glycosyltransferase M.
truncatula UGT72L1, which can produce epicatechin
3'-O-glucoside.
In alternative embodiments, the UGT is the glycosyltransferase M.
truncatula UGT71G1, which uses UDP-glucose as a sugar donor, and is
a multifunctional glycosyltransferase, and has as substrates (can
glycosylate) quercetin, genistein, biochanin A, hederagenin, and
SN-38 (active metabolite of anti-cancer drug CPT-11 or irinotecan),
and (iso)flavonoids such as quercetin and genistein are preferred
in vitro substrates.
In alternative embodiments, the UGT is the glycosyltransferase
UGT85H2, which utilizes UDP-glucose as a donor, and which is a
multifunctional (iso)flavonoid glycosyltransferase with activity
toward several flavonoid-related secondary metabolites, including
isoflavones, flavonols, and chalcone, and has as substrates
quercetin; genistein; biochanin A, kaempferol, and
isoliquiritigenin.
In alternative embodiments, the UGT is the glycosyltransferase
Medicago UGT78G1, which utilizes UDP-glucose as a donor, which is
an (iso)flavonoid glycosyltransferase with broad activities toward
isoflavones such as formononetin and flavonols such as kaempferol,
and also has activity for the anthocyanidins pelargonidin and
cyanidin.
In alternative embodiments, UGT78G1 is used to catalyze a reverse
reaction to remove a sugar moiety, where the enzyme converts
biochanin A 7-O-glucoside, genistein 7-O-glucoside, kaempferol
3-O-glucoside, and quercetin 3-O-glucoside into corresponding
aglycones. In alternative embodiments, UGT78G1 uses quercetin;
genistein; Biochanin A, kaempferol; formononetin, daidzein,
apigenin, myricetin, pelargonidin and cyanidin as a substrate.
In alternative embodiments, the UGT is the glycosyltransferase
UGT88D7, which utilizes UDP-glucuronic acid as a donor, and can use
apigenin, baicalein, scutellarein, kaempferol, quercetin and
naringenin as a substrate.
In alternative embodiments, the UGT is the glycosyltransferase
BpUGT94B1, which utilizes UDP-glucuronic acid as a donor, and can
use cyanidin 3-O-glucoside and cyanidin 3-O-6'-O-malonylglucoside
as a substrate.
In alternative embodiments, the UGT is the glycosyltransferase
UGT89C1, which utilizes UDP-rhamnose as a donor, and can use
kaempferol, kae3-O-glucoside and quercetin 3-O-glucoside as a
substrate.
In alternative embodiments, the UGT is the glycosyltransferase rice
OsCGT, which utilizes UDP-glucose as a donor, and can use
phloretin, 2-hydroxy-eriodictyol and 2-hydroxynaringenin as a
substrate.
In alternative embodiments, the UGT is the glycosyltransferase
buckwheat FeCGTa (UGT708C1), which utilizes UDP-glucose as a donor,
and can use phloretin, 2-hydroxyeriodictyol and 2-hydroxynaringenin
as a substrate.
In alternative embodiments, provided are methods for making
modified or genetically engineered UGTs by manipulating the
acceptor binding pocket of the UGT, or by manipulating multiple
components of the UGT, where the modifications can be by insertion
and deletion, for example, using QUIKCHANGE STRATEGY.TM.
(Stratagene). In alternative embodiments, GT-A, GT-B and GT-C folds
of UGTs are modified or genetically engineered to fit desired
target compounds.
In alternative embodiments, provided are methods for expressing
UGTs, including modified or genetically engineered UGTs, in
bacteria such as E. coli, or in yeast, baculovirus, mammalian
and/or in vitro expression systems. The expressed enzymes can be
then purified or isolated by processes comprising use of
Ni.sup.2+-NTA agarose and SUPERDEX-200.TM. gel filtration, or
equivalents.
In alternative embodiments, the purified or isolated modified or
genetically engineered UGTs are screened for activity on target
compounds such as small molecules, for example, on small molecule
drugs. UDP-glucose, UDP-glucuronic acid and/or UDP-rhamnose can be
used as the sugar donor substrate. In alternative embodiments,
sugar donor specificity is tested. For example, in alternative
embodiments, modified or genetically engineered UGTs are screened
for specificity for UDP-glucose, UDP-glucuronic acid and/or
UDP-rhamnose as the sugar donor molecule. In alternative
embodiments, modified or genetically engineered UGTs are screened
for their ability to modify different small molecules such as
flavonoids or small molecule drug by glycosylation with
UDP-glucose, UDP-glucuronic acid and/or UDP-rhamnose.
In alternative embodiments, LC-MS and NMR analysis are used before,
during and/or after the screening reaction, for example, LC-MS and
NMR analysis are used before purification of the reaction products
from enzyme assays with the bacterial-, yeast- or
baculovirus-expressed modified or genetically engineered UGT. In
alternative embodiments, expressed recombinant or genetically
modified UGT has an altered or new donor and/or acceptor
specificity, and methods as provided herein comprise screening for
the new or altered UGT donor and/or acceptor specificity in a
bacterial, yeast, baculovirus, mammalian or in vitro expression
system. In alternative embodiments, any bacterial, yeast,
baculovirus, mammalian or in vitro expression system can be used,
for example, as described in U.S. Pat. Nos. 9,909,106; 10,017,783;
10,172,931; 10,344,288; 10,294,484; and U.S. patent application
publications nos. 20180127728; 20180087070; 20190092838.
Any of the above aspects and embodiments can be combined with any
other aspect or embodiment as disclosed here in the Summary,
Figures and/or Detailed Description sections.
As used in this specification and the claims, the singular forms
"a," "an" and "the" include plural referents unless the context
clearly dictates otherwise.
Unless specifically stated or obvious from context, as used herein,
the term "or" is understood to be inclusive and covers both "or"
and "and".
Unless specifically stated or obvious from context, as used herein,
the term "about" is understood as within a range of normal
tolerance in the art, for example within 2 standard deviations of
the mean. About can be understood as within 20%, 19%, 18%, 17%,
16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,
1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless
otherwise clear from the context, all numerical values provided
herein are modified by the term "about."
Unless specifically stated or obvious from context, as used herein,
the terms "substantially all", "substantially most of",
"substantially all of" or "majority of" encompass at least about
90%, 95%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of
a composition.
The entirety of each patent, patent application, publication and
document referenced herein hereby is incorporated by reference.
Citation of the above patents, patent applications, publications
and documents is not an admission that any of the foregoing is
pertinent prior art, nor does it constitute any admission as to the
contents or date of these publications or documents. Incorporation
by reference of these documents, standing alone, should not be
construed as an assertion or admission that any portion of the
contents of any document is considered to be essential material for
satisfying any national or regional statutory disclosure
requirement for patent applications. Notwithstanding, the right is
reserved for relying upon any of such documents, where appropriate,
for providing material deemed essential to the claimed subject
matter by an examining authority or court.
Modifications may be made to the foregoing without departing from
the basic aspects of the invention. Although the invention has been
described in substantial detail with reference to one or more
specific embodiments, those of ordinary skill in the art will
recognize that changes may be made to the embodiments specifically
disclosed in this application, and yet these modifications and
improvements are within the scope and spirit of the invention. The
invention illustratively described herein suitably may be practiced
in the absence of any element(s) not specifically disclosed herein.
Thus, for example, in each instance herein any of the terms
"comprising", "consisting essentially of", and "consisting of" may
be replaced with either of the other two terms. Thus, the terms and
expressions which have been employed are used as terms of
description and not of limitation, equivalents of the features
shown and described, or portions thereof, are not excluded, and it
is recognized that various modifications are possible within the
scope of the invention. Embodiments of the invention are set forth
in the following claims.
The invention will be further described with reference to the
examples described herein; however, it is to be understood that the
invention is not limited to such examples.
EXAMPLES
Unless stated otherwise in the Examples, all recombinant DNA
techniques are carried out according to standard protocols, for
example, as described in Sambrook et al. (1989) Molecular Cloning:
A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current
Protocols in Molecular Biology, Current Protocols, USA. Standard
materials and methods for plant molecular work are described in
Plant Molecular Biology Labfax (1993) by R. D. D. Croy, jointly
published by BIOS Scientific Publications Ltd (UK) and Blackwell
Scientific Publications, UK. Other references for standard
molecular biology techniques include Sambrook and Russell (2001)
Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring
Harbor Laboratory Press, NY, Volumes I and II of Brown (1998)
Molecular Biology LabFax, Second Edition, Academic Press (UK).
Standard materials and methods for polymerase chain reactions can
be found in Dieffenbach and Dveksler (1995) PCR Primer: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, and in
McPherson at al. (2000) PCR--Basics: From Background to Bench,
First Edition, Springer Verlag, Germany.
Example 1: Methods and Engineered Enzymes for the Glycosylation of
Bioactive Compounds and Drugs with Various Sugar Donors
Based on Medicago truncatula UGT structures we determined
previously, molecular modeling and docking based enzyme design is
performed to generate novel biocatalysts for glycosylation of drugs
with various sugar donors. In alternative embodiments, UGT88D7 and
BpUGT94B1, which utilize UDP-glucuronic acid, and UGT89C1 which
recognizes UDP-rhamnose, are designed and engineered. UGT71G1
exhibited very broad substrate specificity for glucosylation, and
will also be a mutation template to change its sugar specificity
for other types of sugars, such as UDP-glucuronic acid and
UDP-rhamnose. The newly generated UGT biocatalysts are used for
glycosylation modification of various bioactive compounds and
drugs.
C-Glycosylation of Bioactive Compounds and Drugs
C-glycosides may be more important in pharmacological activity, but
their chemical synthesis is even more difficult. So far, there are
several C-GTs identified in plants, e.g., rice OsCGT, and buckwheat
FeCGTa, and these CGTs only recognize flavonoids. Molecular
modeling is performed and docking based enzyme design is used to
generate novel CGT biocatalysts for glycosylation of various
bioactive compounds and drugs. Also, these C-GTs are engineered to
manipulate their sugar specificity and utilize other types of
sugars for the glycosylation modification of drugs.
Glycosylation is the reaction to transfer sugars from donor
molecules to various acceptor molecules including macromolecules
and small molecules such as natural products. It is a key mechanism
in determining chemical complexity and diversity of small molecular
natural products and bioactive compounds (1). Glycosylation often
enhances solubility, stability and bioactivity, often also reducing
toxicity. Glycosylation can improve bioavailability and
pharmacological activities with implications in drug discovery. So
it is significant and valuable to glycosylate the bioactive
compounds and drugs for drug discovery or reducing drugs' toxicity
or improving their bioactivity and bioavailability.
For example, quercetin is a valuable bioactive flavonoid with
antioxidant activity. Quercetin may be decorated with different
sugars to have different activities. Quercetin-7-glucuronide with
sugar glucuronic acid in 7-OH position is a more efficient
antioxidant (2). Ivermectin is a medication used to treat many
types of parasite infestations, and a glycoside modified with
sugars. Glycyrrhizin is a sweetener, 50-100 times sweeter than
sucrose, and is a triterpene glycoside attached with sugars.
Catechin and epicatechin can be glucuronosylated by mammalian
glucuronosyltransferases to have enhanced brain bioavailability,
and catechin and epicatechin glucuronides were detected in rat
brain and associated with alleviation of the development of
Alzheimers' symptoms (3) and attenuation of features of metabolic
syndrome (4).
Uridine diphosphate glycosyltransferases (UGTs), members of family
1 of the glycosyltransferase superfamily, are the central players
for the glycosylation of small molecules (5). In human, there are
27 UGT sequences identified, and these UGTs are key phase II drug
metabolizing enzymes and play central roles in metabolism and
detoxification of foreign chemicals such as carcinogens and
hydrophobic drugs (6). In plants, a large number of UGTs have
evolved for the glycosylation of plant natural products, e.g. 107
UGTs have been identified in Arabidopsis thaliana (7), and over 300
UGTs are also present in a model legume Medicago truncatula and
other plant species. Glycosylation mediated by UGTs is one of the
major factors determining natural product bioactivity and
bioavailability (8)(9). UGTs have attracted extensive research
interest due to their physiological functions and their potential
application inbiotechnology.
UGT Structures:
We determined crystal structures of three plant UGTs, including
Medicago truncatula UGT71G1, UGT85H2, and UGT78G1 (see FIG. 1A-C)
(10) (11) (12). UGT71G1 is a multifunctional triterpene/flavonoid
glycosyltransferase (13), UGT85H2 and UGT78G1 are (iso)flavonoid
glycosyltransferases (14). They all prefer UDP-glucose as sugar
donor to transfer glucose to small molecules and produce
glucosides. These studies provided the first structural insights
into UGTs and the glycosylation mechanism of small molecules, and
revealed detailed interactions between enzyme and substrates
including sugar donor and acceptor (FIG. 1B-C). Structure-based
mutagenesis studies on UGT71G1 and UGT85H2 showed that we were able
to manipulate the regio-selectivity of glycosylation, and improve
the enzyme activity and quality by site-directed mutations
(15)(16).
FIG. 1A-C illustrate images of crystal structures of three plant
UGTs:
FIG. 1A illustrates the structure of Medicago UGT71G1; the
structure consists of two similar N- and C-terminal domains, and
the active site is located in a deep cleft between the two
domains;
FIG. 1B illustrates the structure of donor binding site and
interaction between the donor molecule UDP-glucose and the enzyme;
a plant UGT specific PSPG signature motif is shown as a ribbon
model in yellow; the structure of UDP-glucose is shown as a
ball-and-stick model;
FIG. 1C illustrates structures comparing acceptor binding pockets
of several plant UGTs including UGT71G1 (dimgrey), UGT78G1 (cyan),
VvGT1 (blue), UGT85H2 (orange), and UGT72B1 (lightgrey); catalytic
residues are in the UGT71G1 structure; residue numbers in UGT71G1
are labeled, and the UGT72B1 unique long loop (approximately
residue 315) is also labeled.
Modification of bioactive compounds and drugs with plant UGTs and
their mutants: The UGT structural studies showed that different
UGTs have quite different sequences in their substrate binding
pockets including the length and composition of amino acids for
recognizing various acceptor substrates (FIG. 1C). We utilize plant
UGTs and their mutants for glycosylation modification of bioactive
compounds and drugs.
Glycosylation modification of bioactive compounds and drugs with
UGT71G1 and other UGTs: UGT71G1 can glycosylate terpenoids (e.g.,
hederagenin), flavonoids (e.g., quercetin), and SN-38 (active
metabolite of anti-cancer drug CPT-11 or irinotecan). Many
terpenoids and flavonoids are bioactive compounds and drugs with
significant benefits for human health. For example, terpenoid
artemisinin is an anti-malarial drug. Taxol is also a terpenoid
compound and the most well-known natural-source cancer drug.
Phenoxodiol is an isoflavonoid derivative and a new oncological
agent.
UGT71G1 has a relatively large acceptor substrate binding pocket
and may recognize many other small molecules. We utilize UGT71G1
and other UGTs, including recombinantly modified forms, as
biocatalysts for glycosylation modification of various drugs.
Design and engineer UGTs as novel biocatalysts for glycosylation of
drugs: We also design UGT mutants by manipulating the acceptor
substrate binding pocket (FIG. 1C), including single mutation on
acceptor binding pocket, multiple mutation, and insertion and
deletion mutation. We generate chimera UGT biocatalysts with an
acceptor substrate binding pocket complementing with target drug
compounds, for glycosylation of various drugs with UDP-glucose.
Mutant generation, expression and purification: In alternative
embodiments, Mutants of UGT71G1 and other UGTs are constructed
using the QUIKCHANGE.TM. (QuikChange) strategy (Stratagene). In
alternative embodiments, all UGTs and their mutants are expressed
and purified according to established methods, e.g., see reference
(10). Briefly, in alternative embodiments, UGTs and their mutants
are expressed in E. coli BL21(DE3) cells. The target proteins can
be purified with Ni.sup.+2-NTA agarose and SUPERDEX-200.TM. gel
filtration column.
Production of glycosylated drugs--Enzyme assays: In alternative
embodiments, enzyme assays are performed essentially according to
the method as described e.g., in reference (10). In alternative
embodiments, the target drugs and bioactive compounds (e.g.,
catechin, epicatechin) are used as substrates for these UGTs and
their mutants. UDP-glucose can be used as sugar donor substrate. In
alternative embodiments, the reaction mixtures are then subjected
to analysis, e.g., LC-MS and NMR analysis, and/or the glycosylated
drugs can be purified.
Production of glycosylated drugs--Bacterial whole-cell UGT mediated
glycosylation: In alternative embodiments, bacterial whole-cell UGT
mediated glycosylation is carried out for glycosylation of drugs,
e.g., according to an established method as set forth in references
(17), (18). In alternative embodiments, E. coli strains carrying
the UGT mutants are grown in Terrific Broth (TB) at 37.degree. C.
When the OD.sub.600 nm of the bacterial culture reaches 0.7, 0.1 mM
substrates, and UDP-glucose are added into the culture followed by
the addition of 0.1 mM isopropyl-1-thio-h-D-galactopyranoside. In
alternative embodiments, the culture is incubated for 24 h at
20.degree. C., the medium and bacterial cells are separated by
centrifugation e.g., for 30 min at 4,000.times.g at 4.degree. C. In
alternative embodiments, the medium is acidified with 6 N HCl to pH
1, extracted twice with ethyl acetate, and dried under nitrogen
gas, and the products can be analyzed e.g., by HPLC.
Glycosylation of Bioactive Compounds and Drugs with Various Sugar
Donors
Modification of Drugs with Various Sugars by Utilizing Novel
UGTs
UGT71G1 utilizes UDP-glucose as sugar donor substrate, UGT88D7 and
BpUGT94B1 utilize UDP-glucuronic acid, and UGT89C1 recognizes
UDP-rhamnose. We obtained UGT88D7, BpUGT94B1 and UGT89C1 by gene
synthesis. These UGTs are utilized to modify drugs with different
sugars, e.g., glucuronic acid and rhamnose.
Design and Engineer UDP-Glucuronic Acid Specific UGT Mutants for
Modification of Drugs with UDP-Glucuronic Acid
Based on Medicago truncatula UGT structures previously determined,
and sequence alignment analysis, UGT88D7 and BpUGT94B1 are
molecularly modeled to generate their three dimensional structural
models, using e.g., the program MODELLER.TM. (19), and energy
minimization and molecular dynamics simulations are performed,
e.g., with the programs CHIMERA (20) and CHARMM (21).
In alternative embodiments, docking studies with bioactive
compounds and drugs are conducted, e.g., manually, e.g., with the
program COOT.TM. (22) or automatically using the program
AUTODOCK.TM. (23) to predict the interactions between enzymes and
the desired substrates, for example, for Lamiales UGT88D7, which
recognizes flavonoids such as apigenin. In alternative embodiments,
mutants are designed with high activity toward target drug
compounds by removing possible stereochemical hindrance and
enhancing their interactions.
UGT88D7 is 33% identical to UGT71G1, and a structural model of
UGT88D7 was generated using UGT71G1 structure; this is also the
best template for UGT88D7 modeling. UDP-glucuronic acid was modeled
according to the conformation and location of UDP-glucose in the
structure of UGT71G1. Epicatechin, an example of target bioactive
compounds, was docketed into the enzyme active site with its 5-OH
appropriately oriented toward the catalytic residue His13 and close
to UDP-glucuronic acid (FIG. 2). The substrate binding pocket
contains several large hydrophobic residues, i.e., Phe 75, Phe 106,
Phe 107, and Phe 136, indicating some potential stereochemical
hindrance problems. This analysis helps us identify potential
residues for mutation to better complement the target bioactive
compounds and drugs, and also enhance the enzyme-ligand
interactions. In alternative embodiments, UGT88D7 mutants are
designed for glucuronidation of the target bioactive compounds and
drugs. Single or multiple amino acids interacting with acceptor
substrates are selected to design mutants for altering pocket
topology, including size and composition.
FIG. 2 illustrates a substrate binding pocket of UGT88D7 docked
with epicatechin and UDP-glucuronic acid (UDP-GA).
BpUGT94B1 is a sugar-sugar/branch forming glucuronosyltransferase
to catalyze glucuronidation of a sugar already attached to
flavonoid such as cyanidin (24). In alternative embodiments,
modeling and docking studies are similarly performed for BpUGT94B1
for design of mutants to produce disaccharide glucuronides of
bioactive compounds and drugs.
Design and Engineer UDP-Rhamnose Specific UGT for Modification of
Drugs with UDP-Rhamnose
UGT89C1 recognizes UDP-rhamnose as sugar donor substrate, and is
mainly active toward flavonoids. In alternative embodiments, using
approaches similar to those as described above for UDP-glucuronic
acid specific UGT modeling and design, UGT89C1 mutants for
glycosylation of target bioactive compounds and drugs with
UDP-rhamnose are designed and generated.
Design and Engineer UGT71G1 for Manipulation of Donor Specificity
Toward Modification of Drugs with Various Sugars
UGT71G1 exhibited very broad substrate specificity for
glucosylation of various bioactive compounds with UDP-glucose, and
will be a major mutation template to change its sugar specificity
for other types of sugars.
Key amino acids and structural features for UDP-glucuronic acid
specific UGT have been reported. Studies of UGT89C1 and
UDP-rhamnose specificity are also reported. In alternative
embodiments, these key amino acids and structural features are
introduced into UGT71G1 to design and generate novel UGT71G1
mutants with sugar donor specificity for UDP-glucuronic acid or
UDP-rhamnose.
In alternative embodiments, modeling and docking studies of UGT71G1
mutants with the target drug compounds are carried out to design
and generate more mutations to allow the target compounds fit the
mutants' substrate binding pocket well. These novel mutants are
designed to recognize new sugar donors and also have activity on
the target drug compounds.
Mutant generation, expression and purification: In alternative
embodiments, mutants of target UGTs are constructed, and the
enzymes are expressed and purified, e.g., using approaches similar
to those as described above, e.g., using a bacterial, a yeast, a
baculovirus or a mammalian cell-based, or in vitro, expression
system.
Production of glycosylated drugs--Enzyme assay: In alternative
embodiments, enzyme assays are performed using approaches similar
to those as described above. In alternative embodiments,
UDP-glucuronic acid and UDP-rhamnose are be used as sugar donor
substrates for UDP-glucuronic acid and UDP-rhamnose specific UGTs,
respectively.
Production of glycosylated drugs--Bacterial whole-cell UGT mediated
glycosylation: Bacterial whole-cell UGT mediated glycosylation are
carried out to produce glycosides, e.g., using approaches similar
to those as described above, e.g., using E. coli or yeast or a
baculovirus systems. In alternative embodiments, for
glucuronidation, an engineered E. coli strain that can accumulate
UDP-glucuronic acid by deleting the araA gene encoding
UDP-4-deoxy-4-formamido-L-arabinose
formyltransferase/UDP-glucuronic acid C-4'' decarboxylase that use
UDP-glucuronic acid as a substrate is used (25). This E. coli
accumulating UDP-glucuronic acid are transformed with the active
glucuronosyltransferase mutants for production of glucuronides.
C-glycosylation of bioactive compounds and drugs: Glycosylation
often occurs on oxygen atoms, UGT71G1 and other target UGTs above
are all involved in the O-glycosylation. C-glycosylation also
occurs, and many C-glycosides are identified, such as C-glycosyl
flavones maysin and apimaysin from corn (26); apigenin
6-C-glucosyl-8-arabinoside (schaftoside), apigenin
6-C-arabinosyl-8-C-glucoside (isoschaftoside), luteolin
8-C-glucoside (orientin) and luteolin 6-C-glucoside (isoorientin)
detected in tea (27); and daidzein 8-C-glucoside (puerarin) from
kudzu. Puerarin has anticonvulsive, antidipsotropic activity and
protective effect on diabetic retinopathy (28). C-glycosides are
very stable since their C--C bonds are resistant to glycosidase or
acid hydrolysis, and may be more important in pharmacological
activity.
Several C-GTs have been identified in several plant species,
including OsCGT from rice (Oryza sativa)(29), GtUF6CGT1 from
Japanese gentian (Gentiana triflora) (30), MiCGT which is a novel
benzophenone C-glycosyltransferase from Mangifera indica(31),
FeCGTa (UGT708C1) and FeCGTb (UGT708C2) from Fagopyrum esculentum
M. (buckwheat)(32), and a 2-hydroxyflavanone C-glucosyltransferase
(UGT708D1) from soybean(33). These C-GTs utilize UDP-glucose as
sugar donor, and catalyze the C-glycosylation of flavonoids. As
described above, some key amino acids and structural features have
been identified, e.g., key arginines (Arg25 in red daisy BpUGT94B1,
Arg350 in Lamiales F7GAT UGT88D7) can interact with anionic
carboxylate of the glucuronic acid as the key requirement for sugar
specificity of UDP-glucuronic acid. We obtained OsCGT and FeCGT by
gene synthesis. In alternative embodiments, these C-GTs are
genetically engineered to change their sugar and acceptor
specificity toward C-glycosylation of drugs.
Manipulation of acceptor specificity for C-glycosylating drugs: In
alternative embodiments, molecular modeling studies of these C-GTs
is conducted using the structures of UGT71G1 and other known UGT
structures as templates. In alternative embodiments, the substrate
binding pocket of these C-GTs are modified to complement the target
drug compounds and to effect the C-glucosylation of drugs.
Manipulation of donor specificity for C-glycosylating drugs: These
C-GTs recognize UDP-glucose for glycosylation. In alternative
embodiments, key amino acids and structural features for
UDP-glucuronic acid or UDP-rhamnose are modified. In alternative
embodiments, C-GT mutants are molecularly modeled and designed to
have different sugar donor specificity such that they can modify
their target drug compounds with different sugars.
Mutant generation and activity assays for production of
C-glycosylated drugs: Rice OsCGT and buckwheat FeCGTa are obtained
by gene synthesis. All C-GT mutants can be constructed using the
QUIKCHANGE.TM. strategy (Stratagene), and expressed and purified
according to established methods. In alternative embodiments,
enzyme assays are performed, e.g., using established methods e.g.,
as described in reference (10). Bacterial whole-cell UGT mediated
C-glycosylation can be carried out to produce C-glycosides as
described herein.
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A number of embodiments of the invention have been described.
Nevertheless, it can be understood that various modifications may
be made without departing from the spirit and scope of the
invention. Accordingly, other embodiments are within the scope of
the following claims.
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